Week 42 (14th-20th of October)

15th of October

1st Western Blot with anti-GFP antibodies

Team members: Noel & Jonas

Materials

ON cultures expressing the construct at question , negative control (no GFP) and positive control (GFP). Here, the analysed cultures are the following: a)GPER-sfGFP b) xLHCGR-sfGFP
MiliQ-water
Lysis Buffer: 50 mM phosphate buffer with 1 mM PMSF
Glass beads
Ice
5 M Guanidinium chloride
Pre-cast SDS gel
5x Protein loading buffer
1x MOPS buffer
Precut Membrane plus filter papers
Low-fat milk powder
PBS-T buffer
Primary antibody: Anti-GFP, rabbit serum
Secondary antibody: Anti-rabbit-HRP, polyclonal swine antibody
A case to keep the membrane
Kit with HRP substrate to make secondary Antibody visible in the gel doc

Procedure

The cultures are spun down
Resuspension in MiliQ-water (1ml)
The cultures are spun down again
Resuspension in 500 µl lysis buffer
Transfer suspension to a 2 ml Eppendorf tube containing 500 µl of Glass beads
Vortex for 1 minute and let chill on ice for 1 minute
Repeat step six nine times
The layer above the glass beads is transferred into a new tube and centrifuged at 4°C (pre-cool centrifuge) for 15 minutes
The supernatant is collected. It contains all the soluble proteins
The pellet is resuspended in 500 µl Guanidinium chloride
The suspension is boiled at 99° C in the heating block and then centrifuged at max speed.
The supernatant is collected. It contains the normally insoluble (membrane) proteins.
For sample preparation 40 µl of protein solution (soluble or insoluble) are mixed with 10 µl 5x Protein loading buffer
A pre-cast SDS gel is put into the gel cassette which is then filled with 1x MOPS buffer
20 µl of the prepared sample are loaded into the wells. A protein pre-stained marker is also loaded in the wells (5µl)
The gel is run at 200 V for one hour
The actual Western Blot is done by assembling membrane, gel and filter papers according to the instruction of the fast blotting apparatus. This Western blot “stack” is put into the fast blotting apparatus.
After protein has been transferred to the membrane, the gel is thrown away and the membrane is put into 25 ml of “PBST milk” ( 5% dry low-fat milk powder in PBST buffer).
The membrane is left on the shaker for at least on 1h at room temperature
The primary antibody is then added directly into the milk. In our case, this was a anti-GFP, rabbit serum antibody. In the end, a 5000x solution should be obtained (5 µl in 25 ml milk)
1-2h incubation at room temperature, alternatively at 4 °C overnight. The membrane is kept shaking
The antibody- milk solution is saved and kept at -20° C for further use
The membrane is now washed three times in PBST-Buffer for at least 5, preferably 15 minutes
After the last washing fluid has been removed, the a 2000x solution of the secondary antibody in 5% PBST milk is added. In our case this antibody is an anti-rabbit-HRP, polyclonal swine antibody (12,5 µl of antibody solution in 25 ml milk).
1-2h incubation at room temperature, alternatively at 4 °C overnight. The membrane is kept shaking
After the incubation, the membrane is washed again three times with 25 ml PBST buffer
Finally, the membrane is transferred to a plastic layer and put into the Geldoc. The two solutions of the visualization kit are mixed and directly pipetted onto the membrane. First, a Picture that allows detection of the light emitted by the HRP reaction is taken. Then a bright field picture is taken to visualize the protein ladder. The two pictures are merged so the protein ladder seen next to the spots on the Western Blot.

Tip from the Supervisors: When washing the membrane with PBST for the first time. Have some “small washing” steps before the actual 5-15 minutes washing steps: Just let a little bit of PBST into the case with the membrane and pour it out a couple of times until the solution that comes out is not “milky” anymore.

Results

Unfortunately, the assay wasn’t successful for the xLHCGPRs due to a systematic error. The positive and negative control have worked as expected. GFP was so strongly expressed in the positive control cells that is was even seen in the insoluble fraction. Vice versa, GPER-sfGFP was predominant in the insoluble fraction, which we expected since it is a membrane protein. A small band can however also be seen in the soluble fraction, indicating that the protein is very abundant in the respective cells.

Figure 1: Western Blot of the soluble fraction of the cells transformed with GPER-sfGFP. The xLHCGR samples between the positive control (GFP) and the samples are cropped out.

Figure 2: Western Blot of the insoluble fraction of the cells transformed with GPER-sfGFP. The xLHCGR samples between the positive control (GFP) and the samples are cropped out.

17th of October

Confocal microscopy

Team members: Jonas

We performed confocal microscopy on postive control (sfGFP yeast), negative control, XLHCGR-sfGFP and GPER-sfGFP samples

Materials

Confocal microscope
Glass slides
Cover slides
Supervisor who is actually allowed to use the confocal microscope
Postive control sample (sfGFP yeast)
Negative control sample
XLHCGR-sfGFP sample
GPER-sfGFP sample

Procedure

The microscope was already turned on so we didn't have to do that
Prepare slides by adding 8 microliter of sample onto glass slide and putting a cover slide on top
Putting slide into the microscope
Finding the correct focus plane so we can actually see the cells
Then we took bright field and flourescence images using the PC software for the confocal microscope

Results

IMAGES ARE MISSING

2. Repetition of the anti-sfGFP Western Blot

Team members: Noel & Jonas

For the first WesternBlot, we accidentally chose the wrong cultures of xLHCGR-sfGFP. Therefore, the result was negative as we expected.

Materials and Procedure

The procedure as described on the entry of the 15th october as repeated.

Results

The results contradicted our expectations as the LH receptor construct only seems to appear in the soluble fraction. Furthermore, it seems to be fragmented as multiple bands can be seen. Finally, it needs to be noted, that the positive controls shows a smear instead of a single band in the soluble fraction. There could have been a problem in the preparation of soluble proteins. We accept this result but take it with a grain of salt. But anyway it is notable that the xLHGPER appears in the soluble but not in the insoluble fraction.